Proteases from granulocytes and macrophages have been reported to be responsible for the chronic tissue destruction mechanisms associated with inflammation, including rheumatoid arthritis and emphysema. Accordingly, specific and selective inhibitors of these proteases are candidates for potent anti-inflammatory agents useful in the treatment of inflammatory conditions resulting in connective tissue destruction, e.g. rheumatoid arthritis, emphysema, bronchial inflammation, chronic bronchitis, glomerulonephritis, osteoarthritis, spondylitis, lupus, psoriasis, atherosclerosis, sepsis, septicemia, shock, myocardial infarction, reperfusion injury, periodontitis, cystic fibrosis and acute respiratory distress syndrome.
The role of proteases from granulocytes, leukocytes or macrophages are related to a rapid series of events which occurs during the progression of an inflammatory condition:                (1) There is a rapid production of prostaglandins (PG) and related compounds synthesized from arachidonic acid. This PG synthesis has been shown to be inhibited by aspirin-related nonsteroidal anti-inflammatory agents including indomethacin and phenylbutazone. There is some evidence that protease inhibitors prevent PG production;        (2) There is also a change in vascular permeability which causes a leakage of fluid into the inflamed site and the resulting edema is generally used as a marker for measuring the degree of inflammation. This process has been found to be induced by the proteolytic or peptide cleaving activity of proteases, especially those contained in the granulocyte, and thereby can be inhibited by various synthetic protease inhibitors, for example, N-acyl benzisothiazolones and the respective 1,1-dioxides. Morris Zimmerman et al., J. Biol. Chem., 255, 9848 (1980); and        (3) There is an appearance and/or presence of lymphoid cells, macrophages and polymorphonuclear leukocytes (PMN). It has been known that a variety of proteases are released from the macrophages and PMN, further indicating that the proteases do play an important role in inflammation.        
In general, proteases are an important family of enzymes within the peptide bond cleaving enzymes whose members are essential to a variety of normal biological activities, such as digestion, formation and dissolution of blood clots, the formation of active forms of hormones, the immune reaction to foreign cells and organisms, etc., and in pathological conditions such as the degradation of structural proteins at the articular cartilage/pannus junction in rheumatoid arthritis etc.
Elastase is one of the proteases. It is an enzyme capable of hydrolyzing the connective tissue component elastin, a property not contained by the bulk of the proteases present in mammals. It acts on a protein's nonterminal bonds which are adjacent to an aliphatic amino acid. Neutrophil elastase is of particular interest because it has the broadest spectrum of activity against natural connective tissue substrates. In particular, the elastase of the granulocyte is important because, as described above, granulocytes participate in acute inflammation and in acute exacerbation of chronic forms of inflammation which characterize many clinically important inflammatory diseases.
Proteases may be inactivated by inhibitors which block the active site of the enzyme by binding tightly thereto. Naturally occurring protease inhibitors form part of the control or defense mechanisms that are crucial to the well-being of an organism. Without these control mechanisms, the proteases would destroy any protein within reach. The naturally occurring enzyme inhibitors have been shown to have appropriate configurations which allow them to bind tightly to the enzyme. This configuration is part of the reason that inhibitors bind to the enzyme so tightly (see Stroud, “A Family of Protein-Cutting Proteins” Sci. Am. Jul. 1974, pp. 74-88). For example, one of the natural inhibitors, .alpha.sub.1-Antitrypsin, is a glycoprotein contained in human serum that has a wide inhibitory spectrum covering, among other enzymes, elastase both from the pancreas and the PMN. This inhibitor is hydrolyzed by the proteases to form a stable acyl enzyme in which the active site is no longer available. Marked reduction in serum α1-antitrypsin, either genetic or due to oxidants, has been associated with pulmonary emphysema which is a disease characterized by a progressive loss of lung elasticity and resulting respiratory difficulty. It has been reported that this loss of lung elasticity is caused by the progressive, uncontrolled proteolysis or destruction of the structure of lung tissue by proteases such as elastase released from leukocytes. J. C. Powers, TIBS, 211 (1976).
Applicants have surprisingly discovered that inhibitors of neutrophil elastase, and in articular, elastase inhibitors of Formula I, may be useful in the treatment of severe neutropenia, especially, cyclic neutropenia (CN) or severe congenital neutropenia (SCN) attributable to mutations in the neutrophil elastase (NE, ELA2) gene.
Severe congenital neutropenia (SCN) is a hematopoietic disorder characterized by maturation arrest at the promyelocytic stage of differentiation, recurring infections, and evolution to acute myeloid leukemia. Mutations in either the neutrophil elastase (NE) gene (sporadic or autosomal dominant SCN) or in the HAXI gene (sporadic or autosomal recessive SCN) lead to a similar clinical phenotype and similar morphological changes of “maturation arrest” in the marrow. Most studies now indicate that maturation arrest in SCN is attributable to accelerated apoptosis of myeloid progenitor cells triggered by the mutant gene products. Cyclic neutropenia (CN) is a hematopoietic disorder also characterized by recurring severe infections and regular oscillations of blood neutrophils from zero to near normal level. These patients also have mutations in the neutrophil elastase gene and also exhibit accelerated apoptosis of bone marrow myeloid progenitor cells.
We have established a model of SCN with doxycycline-regulated expression of del.145-152 mutant NE in human promyelocytic tet-off HL-60 cells. The ratio of normal-to-mutant NE products is approximately 1:1, which is expected in SCN patients with heterozygous mutation. Expression of mutNE in promyelocytic cells resulted in a block of myeloid differentiation with ˜70% reduction in differentiated neutrophils similar to that observed in SCN. The reduced cell growth and accelerated apoptosis were also observed in response to mutNE expression. Thus, this cellular model of SCN appears to closely recapitulate the human phenotype. The elastase-specific activity in cells expressing mutNE was approximately 40% higher than in control cells suggesting that mutNE exhibits at least some proteolytic activity. Screening this SCN model with various agents revealed a cell-permeable proprietary NE-specific small molecule inhibitor, Compound 242, disclosed hereinunder, which inhibited the NE activity by more than 80%. Data thus far indicate that treatment of HL-60 cells expressing the del.145-152 mutNE with Compound 242 led to complete restoration of cell growth and increased myeloid differentiation in these cells. Inhibition of NE activity did not reduce the growth rate of control cells expressing normal elastase. These data confirm our belief that the NE-specific inhibitor Compound 242 and the other compound of Formula I as disclosed hereinunder are useful for the treatment of patients with SCN or CN attributable to mutant NE.